The Drosophila eye is an excellent model system in which to study signaling pathways that are involved in the development of the human eye as well as in other developmental and disease contexts. A genetic mosaic screen has been used to identify novel genes required for the normal pattern of photoreceptor differentiation in the eye disc. This proposal will investigate several such genes that are required for normal activity of the Epidermal growth factor receptor (EGFR) signaling pathway, which controls the proliferation, survival and differentiation of retinal progenitor cells and is misregulated in many human cancers. One such gene, mago nashi, encodes a component of the exon junction complex (EJC), which is deposited onto all spliced mRNAs. Mutants lacking Mago nashi or other EJC subunits show a specific and dramatic reduction in the levels of the mRNA encoding Mitogen-activated protein kinase (MAPK), an essential downstream component of the EGFR and other receptor tyrosine kinase signaling pathways. The first aim of this proposal is to determine whether the EJC is required for normal mapk pre-mRNA splicing, or regulates the stability of the mature mRNA. Either mechanism would represent a novel mode of action for this poorly understood complex, and would contribute to our understanding of the regulation of MAPK expression. Two additional genes required for photoreceptor differentiation encode endosomal proteins, Myopic and Vps4;the second aim of this proposal will seek to understand the role of endocytosis in signaling by the EGFR and other receptors. In myopic mutant cells, EGFR protein accumulates but is unable to signal. The mechanism by which Myopic enhances EGFR signaling will be determined. The possible role in transcriptional regulation of a cleavage fragment of the EGFR formed within the endocytic pathway will also be investigated. The mutation in Vps4, which acts at the final step of protein sorting into multivesicular bodies for degradation, will be used as a tool to determine how receptor activity is altered during this sorting step. The third aim will seek to clone and characterize two novel genes found in a screen of the X chromosome that are likely to affect signaling through the EGFR pathway. ayn and cassandra have been mapped to small chromosomal regions and will be identified by sequencing candidate genes. Biochemical methods will be used to determine their roles within each signaling pathway. Finally, mutations affecting CSN1b, a subunit of the COP9 signalosome, show a dramatic increase in the expression of the EGFR target gene argos. The hypothesis that this change is mediated by alterations in transcription factor ubiquitination will be investigated. Taken together, the proposal will reveal new molecular mechanisms in a conserved signaling pathway that is critical for retinal development. PUBLIC HEALTH RELEVANCE: Retinal development is regulated by a conserved set of signaling pathways. Drosophila genetics offers a powerful, rapid and economical method to identify novel components required for this process and to understand their molecular mechanisms. We propose to characterize new genes required for signaling by the Epidermal growth factor receptor, which is essential for photoreceptor differentiation and the misregulation of which is a major cause of cancer. Evolutionary conservation suggests that our findings will be applicable to higher organisms, including humans, and may lead to tools for diagnosis or treatment of genetic disorders affecting the development of the eye or other organs, or to new cancer therapies.